Project Summary In both humans and mice, natural killer (NK) cells are required for host defense against viral infection, most notably herpesvirus infections. To support viral clearance, NK cells robustly proliferate in an antigen-specific fashion, generate immunological memory, and exhibit enhanced functionality upon secondary encounter with the same virus. However, the molecular mechanisms governing these dynamic NK cell responses to infectious pathogens are incompletely defined. To address the requirements for NK cell expansion, I have examined by RNA sequencing (RNA-seq) the transcriptome of virus-specific NK cells during a model of mouse cytomegalovirus infection (MCMV). By comparing highly expressed genes in proliferating NK cells during MCMV infection to those in nave NK cells, I have identified the transcription factor interferon regulatory factor 8 (IRF8) as being enriched in MCMV-responding NK cells. Preliminary studies revealed that NK cells lacking IRF8 are impaired in their proliferative capacity, and this coincides with host susceptibility to MCMV infection. Thus, the goal of this proposal is thus to define the role of IRF8 in MCMV-specific NK cells during infection. In further preliminary studies, Zbtb32 was identified by RNA-seq as being significantly downregulated in Irf8-/- NK cells versus wild-type (WT) NK cells during MCMV-driven proliferation. This study will first examine whether IRF8 binds regulatory elements within the Zbtb32 locus by chromatin immunoprecipitation and whether overexpressing Zbtb32 in Irf8-/- NK cells, through generating novel retrogenic mice by retroviral transduction, can restore normal MCMV-driven proliferation (Aim 1). Subsequently, this study will use immunoprecipitation and mass spectrometry to identify distinct IRF8 binding partners in activated versus nave NK cells that may underlie the infection-specific transcriptional program of IRF8 (Aim 2). Together, these studies will shed light on how IRF8 induction promotes the generation of potent MCMV-specific NK cell responses. By advancing basic knowledge of the mechanisms governing antiviral NK cell responses, the proposed research may reveal novel strategies for manipulating NK cell responses against pathogens in the clinic.